Variant LovD polypeptides and their uses

Abstract

The present disclosure provides acyltransferases useful for synthesizing therapeutically important statin compound.

1. An isolated or recombinant variant LovD polypeptide having at least two-fold greater acyltransferase activity than the wild-type  Aspergillus terreus  acyltransferase of SEQ ID NO:2, which comprises the amino acid sequence of SEQ ID NO:2 that includes the mutations L174F and A178L and from 1 to 30 additional mutations. 
     
     
       2. The variant LovD polypeptide of  claim 1 , which has at least 10-fold greater acyltransferase activity than the wild-type  A. terrus  acyltransferase of SEQ ID NO:2. 
     
     
       3. The variant LovD polypeptide of  claim 1 , that includes the following additional mutations: A123P, N191S/G, A247S and L361M. 
     
     
       4. The variant LovD polypeptide of  claim 1 , wherein the 1 to 30 additional mutations are selected from the group consisting of 14N, A9V, K26E, R28K, R28S, 135L, C40A, C40V, C40F, C40R, S41R, N43R, N43Y, C60F, C60R, C60Y, C60N, C60H, D96R, S109C, A123P, S142N, A184T, A184V, N191S/G, Q241M, A247S, D254E, A261H, A261T, A261E, A261V, L292R, Q295R, Q297E, L335M, L361M, A377V, A383V, N391D, H404K, H404R, Q412R. 
     
     
       5. The variant LovD polypeptide of  claim 3 , further comprising one or more additional mutations selected from: A9V, K26E, M157V, L192I, R250K, G275S, Q297E/G, and A383V. 
     
     
       6. The variant LovD polypeptide of  claim 5 , wherein the one or more additional mutations are selected from: A9, K26E, and G275S. 
     
     
       7. A method of making simvastatin comprising contacting monacolin J substrate with a variant LovD polypeptide according to  claim 1  in the presence of an α-dimethylbutyryl thioester co-substrate and under conditions in which the monacolin J is converted to simvastatin. 
     
     
       8. The method of  claim 7 , wherein the monacolin J is a sodium salt. 
     
     
       9. The method of  claim 8 , wherein the monacolin J is a sodium salt and activated charcoal is added. 
     
     
       10. The method of  claim 9 , wherein the monacolin J is an ammonium salt. 
     
     
       11. The method of  claim 7 , wherein an agent for precipitating simvastatin is added. 
     
     
       12. The method of  claim 11 , wherein said agent is ammonium hydroxide. 
     
     
       13. A method of making simvastatin comprising contacting a lovastatin substrate with a variant LovD polypeptide according to  claim 1  in the presence of an α-dimethylbutyryl thioester co-substrate and under conditions in which the lovastatin substrate is converted to simvastatin. 
     
     
       14. The method of  claim 13 , wherein said lovastatin substrate, said variant LovD polypeptide and said thioester are charges at substantially the same time into a vessel. 
     
     
       15. The method of  claim 13 , wherein said lovastatin substrate and said variant LovD polypeptide are first charged into a vessel and then said thioester is charged into said vessel. 
     
     
       16. The method of  claim 13 , wherein an agent for precipitating simvastatin is added. 
     
     
       17. The method of  claim 16 , wherein said agent is ammonium hydroxide.